【作者】 刘小丰；

【导师】 林兴华；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2006， 硕士

【摘要】 由水稻黄单胞杆菌（Xanthomonas oryzae pv.Oryzae，简称Xoo）引起的水稻白叶枯病是是一种严重的水稻细菌性病害，目前已经鉴定出约30个白叶枯病抗性基因，其中Xa1、xa5、Xa21、Xa26、Xa27被分离和克隆出来。Xa14是高抗Xoo菲律宾生理小种5（P5）的显性基因，1987年Taura等将其定位在水稻第4染色体长臂末端，1998年谭震波等将其定位于标记RG620和G282之间，2004年本室鲍思元等将其定位在HZR970-8和HZR988-1之间。本研究的目的是对Xa14进一步精细定位，并对鲍思元等定位的区域内与抗性相关基因进行遗传和功能分析，主要结果如下： 1．构建包含3300个单株的IRBB14／IR24的F₂群体，用14对在两亲本间具有多态性的SSR标记将Xa14定位在HZR970-8和HZR669-2之间，并与标记HZR645-4共分离。完全覆盖nipponbare的BAC克隆OSJNBb0085C12和BAC克隆OSJNBa0053K19，部分覆盖BAC克隆OSJNBa0058K23和BACOSJNBB0060E08，约324kb的距离。 2．用FGENESH和GENESCAN对标记HZR970-8和HZR988-1之间的DNA序列进行分析，与F.QI等的预测结果进行比较，筛选含有与抗性有关保守结构（如NBS，LRR，Kinase等）的基因。经过筛选，共得到6个含有NBS结构的候选基因，成簇分布在BAC克隆OSJNBb0085C12上，按排列顺序分别命名为RGA1、RGA5、RGA6、RGA7、RGA12和RGA14。 3．提取抗病亲本IRBB14和感病亲本IR24接种病菌后的总RNA，反转录。对这6个抗性相关基因分别设计特异引物，PCR验证，发现RGA1和RGA5在两个亲本中表达，但RGA12仅在IRBB14中表达。在未接种条件下，剪取材料IR24、IRBB14、MDJ和TP309正常生长时的健康叶片，分别提取RNA，RT-PCR验证RGA1和RGA5在IR24、IRBB14、MDJ和TP309都为组成型表达；RGA12在IRBB14、MDJ和TP309为组成型表达，在IR24中不表达。将这3个基因确定为Xa14的候选基因。 4．对这3个侯选基因设计引物，进行长片段扩增，得到三个候选基因的全长序列，经TA克隆，大肠杆菌增殖后，经质粒操作连接到表达载体pCAMBIA1301S中，电击入农杆菌。经农杆菌转化，将候选基因转入感病材料台北309和珍珠矮中，再生，获得再生植株，实验室考验后移入大田种植。 5．在苗期对3个候选基因的转化植株进行PCR鉴定，保存阳性单株。孕穗期接种鉴定，未能发现抗病能力较转化材料有明显增强的单株。每个转化材料随机抽取8株阳性单株进行SOUTHERN杂交验证，杂交结果证明转化是成功的。实验结果表明RGA1、RGA5和RGA12中不包含Xa14。更多还原

【Abstract】 Bacterial blight, caused by Xanthomonas oryzae pv. Oryzae, is one of the most serious disease of rice worldwide. About thirty bacterial blight resistance gene in rice have been identified, five of them, Xa1, xa5, Xa21, Xa26 and Xa27 have been cloned successfully by a map based cloning approach. Xal4 is a dominant resistance gene against Philippines race 5 of bacterial blight pathogen Xoo, Taura et al. （1987） located it to the distal end on the long arm of chromosome 4, Tang et al （1998） located it between marker RG620 and G282 by RFLP markers, Bao et al. （2004） constructed a high-resolution linkage maps for Xal4 region by SSR markers, and located it between marker HZR970-8 and HZR988-1. In this paper, we had fine mapping Xal4 farther and analysis the sequence structure, expression and function of the resistance gene analogs in the Xa14 region.1. F₂ population consisting of 3300 individuals derived from a cross between IR24 and IRBB14 were screened for recombination events by 14 pair polymorphous SSR markers. Xal4 was located in a 324 kb region, flanked by the markers HZR970-8 and HZR669-2, cosegregated with HZR645-4, covered with nipponbare BAC clones OSJNBb0085C12 and OSJNBa0053K19 completely, OSJNBa0058K23 and BAC OSJNBB0060E08 partly.2. FGENESH and GENES CAN was used to analysis the nipponbare chromosome 4 DNA sequence of markers HZR970-8 and HZR988-1, a BLASTX analysis tool, CCD v2.06 was used to predict protein structure and function. BLASTX analysis showed that only 6 predicted resistance gene analogs had sequence homology with known plant R gene. They all encoded NBS-LRR proteins and clustered together to spread on nipponbare BAC OSJNBa0058K12, This 6 predicted genes were designated RGA1, RGA5, GRA6, RGA7, RGA12 and RGA14.3. Extract total RNA from the undamaged fresh leaves of IR24 and IRBB14 inoculated with PXO112 or water to investingate the expression of the six resistance gene analogs by RT-PCR, result showed RGA1 and RGA5 expressed in two parents, RGA12 only expressed in the resistant parent IRBB14. Extract total RNA from the undamaged fresh leaves of IR24, IRBB14, TP309 and MDJ, RT-PCR analysis indicated that RGA1 and RGA5 had constitutively expressed in four rice materials, RGA12 hadn’t expressed in IR24, but constitutively expressed in other three rice materials. So we selected RGA1, RGA5 and RGA12 as the candidate genes of Xa14. The 3’ ends of the更多还原